Synthesis of a HDC-GFP Promoter-Gene Fusion as a Tool to Identify Histaminergic Cells and Examine HDC Regulation

The promoter region for the Histidine decarboxylase gene (pHdc), which is required for synthesis of the neurotrans-mitter histamine, has been functionally identified in Drosophila melanogaster. A fusion between pHdc and the enhanced Green Fluorescent Protein (eGFP) has been made in a plasmid that will allow generation of transformant flies. These transgenic flies containing the pHdc-eGFP gene fusion can be studied to determine whether the pHdc region causes expression of enhanced Green Fluorescent Protein (eGFP) in hista-minergic cells. This research will allow further analysis of the HDC promoter and be a useful tool for examining the physiology of histaminergic cells. Introduction Histamine is a biogenic amine involved in local immune responses, and it acts as a neurotransmitter (Brown et al., 2001). Histamine is synthesized by the decar-boxylation of the amino acid histidine, catalyzed by the enzyme histidine decarboxylase. In the fruitfly Drosophila melanogaster, histamine is utilized as a functional neurotransmitter for photore-ceptor and mechanosensory cells, but has also been localized by immunocytochem-istry to a few specific cells in the brain The Hdc gene, which codes for this enzyme, has been identified and studied in Drosophila, a model organism well-suited for genetic studies. Mutations in the Hdc gene have been isolated which disrupt function, indicating what the absence of histamine does to the organism. Hdc mutations cause flies to be functionally blind, in addition to other sensory alterations (Burg et al., 1993; Melzig et al., 1996). To further investigate the regulation of the Hdc gene and how expression may be altered throughout development, the Hdc promoter region was identified through a combination of sequence analysis, deletion mapping, and 5' RACE experiments (Sambrook et al., 1989). Through the analysis of deletions constructed in the Hdc promoter region, it was possible to separate the expression of Hdc in photoreceptors from expression in the brain (Burg and Pak, 1996). Recent experiments have identified novel 5' ends of the Hdc cDNA which do coincide with the deletion analysis results, suggesting that the transcriptional promoter for brain Hdc expression is unique from the identified photoreceptor Hdc promoter (M. Burg and S. Smolin-ski, pers.commun.). While deletions in a promoter region can identify regions necessary to guide expression in various tissue types, they cannot identify areas that are sufficient alone for Hdc expression. One method that has been used extensively in promoter analysis studies in Drosophila